Simultaneous removal of endotoxins, inflammatory mediators and uremic toxins in ICU patients with septic shock: a retrospective cohort study

Sepsis, one of the leading causes of death, is still lacking specific treatment. OXIRIS (BAXTER, Deerfield, IL, USA) is the first device allowing combined removal of endotoxins, inflammatory mediators and uremic toxins, alongside fluid balance control. Available data is very limited. This retrospective propensity score-matched cohort study of adult patients with septic shock aimed to evaluate septic shock duration and mortality in patients treated with either standard of care renal replacement therapy (RRT) or RRT with combined hemoadsorption, who were admitted to the interdisciplinary surgical intensive care unit at Heidelberg University Hospital during the years 2018 through 2021. Main outcomes were duration of shock, thirty-day mortality and plasma interleukin-6 levels before and after initiation of hemoadsorption. Included were 117 patients (female, 33%; male 67%); median age: 67 (16) years. After matching: 42 patients (female, 33%; male, 67%); mean age: 59.1 ± 13.8 years. There was no statistically significant difference in septic shock duration (p = 0.94; hazard ratio (HR) 0.97 (95% CI, 0.48–1.97)). Thirty-day survival analysis showed a non-statistically significant survival difference. (p = 0.063; HR 0.43 (95% CI, 0.17–1.09)). A post-hoc 90-day survival analysis revealed statistically significant longer survival and lower death hazard ratio in patients treated with RRT + HA (p = 0.037; HR = 0.42 (95% CI, 0.18–0.99). In conclusion, RRT with combined hemoadsorption of endotoxins, inflammatory mediators and uremic toxins is a modality worth further investigation.

IL-6, secreted by T-cells and a stimulator of antibody secretion by B-cells, plays an important role in the so-called "cytokine storm" present in sepsis 16 .IL-6, IL-10 and TNF-alpha are among the mediators principally augmented during this process 21 .Increasing levels of IL-6 were found to be linked with increased mortality, while lower levels were related with recovery in septic shock 22 .
There is no recommendation for routine treatment of septic shock patients with a hemoadsorptive device 35,36 , and there is a necessity for studies to further elucidate these applications 35 .

Objectives
The aims of this study were to collect data about patients with renal replacement therapy (RRT) ± hemoadsorption (HA) using OXIRIS at our institution and to perform a retrospective explorative analysis.
The main study goal was defined as the duration to resolution of septic shock, as defined by the Sepsis-3 criteria, i.e.: need of vasopressors to achieve a mean arterial pressure (MAP) of ≥ 65 mmHg, and serum-lactate > 2 mmol/L (18 mg/dL) 1 .For the purpose of this study, the time to resolution of septic shock was defined as the time from onset of septic shock until a reduced vasopressor need had been achieved for ≥ 6 h (in terms of a norepinephrine-dosage of < 0.2 µg/kg/min lean body weight (Boer) 37 ), as well as serum-lactate < 2 mmol/L (18 mg/dL).This was considered as a resolution of refractory septic shock.

Participants
Inclusion criteria were: age ≥ 18 years, septic shock as defined by Sepsis-3 criteria 1 .

Data sources
Local electronic patient charts (COPRA, COPRA System GmbH, Berlin, Germany; LAURIS, NEXUS SWISSLAB GmbH, Berlin, Germany and SAP, Walldorf, Germany) were reviewed and data collection was done using MICROSOFT EXCEL (MICROSOFT, Redmond, WA, USA).Subsequent analysis was done using IBM SPSS STATISTICS 28.0.(IBM, Armonk, NY, USA).

Data collection
The following data were collected: Age at time of onset of septic shock, sex, height, weight, body mass index (BMI), lean body weight (BOER), infection focus, prior medical conditions (history of cardiovascular disease [coronary heart disease, arterial hypertension, heart failure, cardiomyopathy, heart valve disease or vitium, arrhythmia, myocardial infarction, heart transplant], kidney failure, history of tumor disease, diabetes), history of smoking, history of alcohol consumption, allergy to heparin or HIT, Sepsis related organ failure (SOFA) scores, Acute Physiology and Chronic Health Evaluation (APACHE) scores, and Simplified Acute Physiology II (SAPS II) scores at time of admission to the ICU.Furthermore, the following were noted: times of onset of septic shock (earliest available documentation in medical records), treatment by RRT ± HA, other adsorptive treatments, initiation-and conclusion-times of RRT ± HA, most recent available plasma IL-6 levels within 24 h before initiation of HA and latest available levels after initiation of HA (up to 24 h of conclusion of HA), time of septic shock resolution (as defined), time of death, administration of other vasopressors than norepinephrine at either time of shock-resolution or death, time of last follow-up at our institution, suspected foci and blood culture results.Intervals were calculated between the times of interest.During data collection, times were noted with precision of up to 15 min-intervals.Times of death were noted as stated in medical records.In case patients had suffered multiple episodes of septic shock, the first such episode was considered, as well as the first-time employment of OXIRIS.Furthermore, data on the conditions for RRT and RRT + HA were collected and described.

Statistics
The study was conceived as a monocentric, retrospective study.Statistics are reported following the SAMPL guidelines 39 .

Explorative analysis and evaluation of homogeneity
An explorative evaluation of data was performed.Descriptive statistics were generated for the overall study population, and differences between groups were compared to find possible differences.Normally distributed continuous variables are reported as mean ± standard deviation (SD); non-normally distributed variables as median (Interquartile Range, IQR)).In categorical variables, "n "-numbers and percentages are reported.
In continuous variables, normal distribution was verified using the Shapiro-Wilk test.Variables with normal distribution were tested using the t-test for independent variables, and the Mann-Whitney-U test was used in skewed data.Differences in proportions were verified using Fisher's exact test considering the restricted patient numbers.Standardized mean differences (SMD) are reported for all variables.Small, medium, and large effect sizes were considered to be represented by SMDs of 0.2, 0.5, and 0.8, respectively 40,41 .
All reported tests are two-tailed and exact significance is reported.Significance levels were defined at α = 0.05.Since both groups were not homogenic across all baseline characteristics, a one-to-one (paired) propensity score matching was performed.

Missing data
Missing data are reported as "n (%)" and patients with missing data were excluded from tests.

Propensity score matching
Concerning the details of propensity score matching (PSM), we kindly refer to the literature [42][43][44] .Rosenbaum and Rubin 44 defined the propensity score as the probability of allocation to a treatment dependent of observed baseline covariates.It summarizes all covariates into one variable describing the probability of having been subjected to the treatment 45 .Distribution of covariates defining the propensity score is the same in treated and control groups for each value in the score. 45Patients with an identical score have the same distribution of measured baseline covariates 42 .The estimated propensity score is usually calculated using a logistic regression model, regressing the treatment modality on monitored baseline covariates 42 .
Before matching, variables describing cardiovascular disease were recombined into a single dichotomous variable for presence or history of cardiovascular disease, defined as present if the patient had a history of any of the following: coronary heart disease, arterial hypertension, heart failure, cardiomyopathy, heart valve disease, arrhythmia, myocardial infarction, or history of heart transplant.
Patients were matched using: SOFA scores, age, history of cardiovascular disease, and history of tumor disease.These were chosen as a compromise of highly suspected influencing factors for treatment assertation of patients, and of the constraint of using a limited number of covariates given the restricted study population 45 .Variable selection was done without analyzing any outcomes 45 .Several matching model iterations were performed, and judged through balance and remaining sample size 46 .Assessment of the resulting model was done before proceeding to the analysis of outcomes 45 .Care was taken as to not lose any patients of the intervention group, considering low numbers.Best results (i.e., remaining sample size and appropriate balance 46 ) were obtained using nearest neighbor caliper matching without replacement, with priority to exact matching, in random order, discussed by Austin et al. 43 Caliper width was set to 0.15.Within the matched sample, comparisons of treatment effects may directly be made 42 .
As recommended by Austin et al. 42 , paired tests were used in the matched sample: paired t-tests for normally distributed continuous variables and the Wilcoxon-test for non-normally distributed continuous variables; McNemar's test for differences in proportions in dichotomous variables.

Primary and secondary outcomes
Survival analysis and hazard ratios.For the Kaplan-Meier analysis 47 regarding duration of septic shock, the time to event period was defined as the duration from onset of septic shock until resolution of refractory septic shock as defined earlier.Patients who died before resolution of refractory septic shock occurred were right-censored.Concerning survival analysis for 30 day-, respectively post-hoc 90 day-mortality, the time to event period was defined as the duration from onset of septic shock to death.Patients alive after 30, respectively 90 days, or who were lost to follow-up within the 30-/90-day period were right-censored.The assumptions of the Kaplan-Meier estimator were met 48 , i.e. one can expect censoring to be independent of the probability of developing the event of interest; furthermore, there is no reason for suspecting different survival probabilities at different times of the observation period.Survival curves were created for the RRT and RRT + HA groups.Results are reported as mean (SD) and median (IQR) with 95% confidence intervals (CI).Statistical significance was tested using the Log-Rank test (Mantel-Cox).
Univariate Cox regression 49 , as discussed by Austin 50 was performed to estimate hazard ratios (HR).The time to event period was defined as the duration from onset of septic shock until resolution of refractory shock (primary outcome) or until death during the 30-/90-day follow-up after onset of septic shock (secondary outcome) and the treatment groups as binary predictor.HRs are reported with 95% CI.
Interleukin-6.To assess the evolution of IL-6 levels before and after initiation of HA, Wilcoxon's signed-rank test was used for patients where IL-6 levels had been measured as described above.For patients with RRT without HA, regrettably IL-6 levels had not been measured during hospitalization and thus were not available.

Population
Screening identified 155 patients.Eleven patients were excluded since RRT was not applied during septic shock.Twenty-seven patients were excluded due to confounding treatments, HIT, or insufficient data.(Fig. 1).Eventually, 117 patients were included in the study.

Propensity score matching
Propensity score matching (PSM) resulted in a collective of 42 patients.The model allowed all patients treated with HA to be matched.

Treatment initiation
Several patients were already treated with some form of RRT before the onset of septic shock (RRT, n = 9/21; RRT + HA, n = 3/21).For patients with standard of care RRT in whom RRT was only initiated after the onset of septic shock (n = 12/21), the median duration from onset of shock to initiation of RRT was 39.3 (34.4) hours.Conversely, patients with RRT + HA in whom RRT was only initiated after the onset of septic shock (n = 18/21), the median interval to RRT initiation was 23.8 (21.7) hours, while the median interval from onset of septic shock to initiation of HA therapy was 24.5 (24.4) hours.

Description of RRT procedures
Membranes.RRT with GENIUS 90 was performed as sustained low-efficiency dialysis (SLED/CVVHD) (FRESENIUS MEDICAL CARE, Bad Homburg v. d.Höhe, Hesse, Germany) with FX 50 filters (FRESENIUS MEDICAL CARE, Bad Homburg v. d.Höhe, Hesse, Germany), with an effective surface area of 1.0 m 2 and a fiber internal diameter (wet) of 185 µm.The FX 50 membrane is a polysulfone-based membrane called HELIXONE (FRESENIUS MEDICAL CARE, Bad Homburg v.d.Höhe, Hesse, Germany).
RRT with PRISMAFLEX was performed with PRISMAFLEX M150 sets (BAXTER, Deerfield, IL, USA) with an effective surface area of 1.5 m 2 and a fiber wall thickness of 50 µm and a fiber internal diameter (wet) of 240 µm.PRISMAFLEX M150 sets include an AN 69 HF hollow fiber (Acrylonitrile and sodium methallyl sulfonate copolymer).
RRT ± HA was performed as CVVHDF using the PRISMAFLEX system (BAXTER, Deerfield, IL, USA).The OXIRIS hollow fiber consists of an acrylonitrile and sodium methallyl sulfonate copolymer and polyethyl-eneImine (surface treatment agent) and is heparin grafted (4500 ± 1500 IU/m 2 ) and has been described in detail above.Due to the retrospective nature of the study, patients unfortunately were not all treated using the same RRT modality.However, both treatment cohorts were relatively comparable in the duration of treatment during septic shock.

Description of SLED/CVVHD
SLED/CVVHD are usually performed with blood flows around 100-150 mL/min with ratio of 1:1 or 2:1 to dialysate flow.

Anticoagulation
In SLED, anticoagulation was usually performed by a continuous infusion of unfractioned heparin, with target PTT levels of 40-60 s, adjustable depending of possible contraindications from the surgeons' or intensivists' point Fig. 2. Distribution of Propensity Scores.showing the distribution of propensity scores before and after propensity score matching, demonstrating an appropriate adjustment of both study groups' propensity scores.

Table 2.
Treatment data for RRT during septic shock.Results are reported as median (IQR).All patients with RRT+HA received CVVHDF with the OXIRIS filter during septic shock.Some patients received either therapy using SLED/CVVHD (FX 50) or CVVHDF (M150) during septic shock before switching to another modality or CVVHDF with OXIRIS.Therefore, the number of patients exceeds 100% of the total patient count.Vol:.( 1234567890)
Missing data for 30-day follow up.Explorative data analysis hinted at some differences in baseline characteristics of patients who were lost to follow-up at 30 days (n = 4/42) compared to patients with 30-day follow-up (n = 38/42), most notably they had a higher median age (71.0 (17.8) vs. 58.5 (19.3)) and higher median BMI

Key results
This retrospective, monocenter study showed no statistically significant differences in the primary outcome parameters duration of refractory septic shock nor thirty-day mortality.A post-hoc 90-day survival analysis showed statistically significant longer survival times and lower death hazard ratio for patients treated with RRT + HA.There was a statistically significant reduction of IL-6 levels after initiation of HA; however, there were no control measurements available.

Generalizability
Due to the retrospective observational nature of this study, patients were not randomly assigned to treatments.Since in retrospective studies patients in treatment arms often diverge systematically from patients in control arms, propensity score matching was employed to balance treatment groups on observed baseline characteristics to attempt to balance systematic differences in both groups 42 .The association of patients with similar propensity scores allows for the replication of a quasi-randomized experiment 45 , imitating some of the characteristics of a randomized controlled trial 42 , and the effect of the treatment may then be compared directly between matched groups 42 .PSM may allow to produce a balance on measured baseline covariates, nonetheless, it is not possible to rule out heterogeneity in unmeasured covariates 42,51 .
Comparison to the literature Mortality.Previous studies have found 90-day mortalities in septic shock of around 39% 52 , crude mortality of about 47% 53 , and 28-/30-day mortality of about 37% 54 , mentioning a substantial heterogeneity of mortality [52][53][54] .For example, Shankar-Hari et al. report a variation from 23% to about 82% 53 .Bauer et al. 52 reported a median age of 64 years, comparable to this study's population median age of around 59 years.The overall 30-day mortality of around 48% (n = 20/42) and 90-day mortality of 55% (n = 23/42) in our matched study population is slightly higher compared to numbers reported in the literature [52][53][54] .The mean SOFA score at time of admission of 12.6 ± 3.3 with a median of 13.0 was substantially higher in our population compared to a median of 9.5 reported by Bauer et al. 52 Septic shock mortality rates were higher in retrospective (around 42%) than in prospective studies 52 .Per SOFA-score increase of one point, 90-day mortality increased by 2.4% 52 .An increase of 3.5 median SOFA score points (13.0 vs. 9.5) would translate into an expected 8.4% increase in mortality to about 47% (from around 39% reported by Bauer et al. 52 .)for this study, approaching our findings.Patients in this study seem to have had higher morbidity considering higher SOFA scores and mortality.www.nature.com/scientificreports/Data on renal replacement therapy and hemoadsorption with OXIRIS is scarce.To the best of our knowledge, only two small randomized prospective studies 18,25 and no studies with PSM have been published 23 .Most studies available on the subject report lower total patient populations.One recent work in 2022 reported comparable numbers, however, no adjustments were made in that case, making it difficult to compare outcomes from our point of view 16 .Interestingly, a recent systematic review and meta-analysis in 2023 found lower seven-, 14-and 28-day mortalities and shorter length of stay in the ICU, but in contrast to the present findings there was no statistically significant difference in 90-day mortality 55 .This meta-analysis however was criticized, among other things, because results from randomized controlled trials and retrospective studies were pooled and because most of the studies lacked detailed reporting 56 .Moreover, half of the considered studies did not report 28-day mortalities and the probable presence of publication bias was commented 57 .
Duration to therapy initiation.The median duration from onset of septic shock to initiation of HA therapy of 24.50 (24.38) hours corresponds well to previously described times of 18-46 h 2,16,19,26 .While the duration to initiation of RRT + HA may seem rather long, this might be due to the fact that treatment with RRT + HA is administered only in select patients, and there is no standard procedure regarding its initiation.One reasonable explanation might be the duration until treatment is approved by a senior clinician.However, one needs to be mindful of these timespans as intervening to remove endotoxins and inflammatory mediators may make the most sense in the very first hours of septic shock.There has to be a careful assessment of benefits and risks for each patient, as there are risks involved with hemoadsorptive procedures and for some devices even negative outcomes have been reported 58 .

Limitations
The number of patients with RRT + HA without exclusion criteria was limited.Regrettably, IL-6 levels were only available for patients with RRT + HA, since these were not measured for patients in the control group during hospitalization.Although both patient cohorts were adequately adjusted through the matching procedure, some differences remained.While less substantial than before matching, this has to be taken into consideration when interpreting the results, as in a small patient cohort even minor differences in characteristics such as past myocardial infarction might interfere with outcomes such as survival, and since patients lost to follow-up were somewhat older.
The fact that patients in the RRT and RRT + HA cohort were not all treated with the same modality or anticoagulants is obviously a clear limitation to this retrospective study.On the other hand, the limitation is relativized due to the lack of data supporting the superiority of any treatment modality in patients with acute kidney injury.Two relatively recent meta-analyses of studies comparing continuous RRT and SLED found no difference in recuperation of kidney function nor mortality 59,60 .A relatively recent meta-analysis on anticoagulants in continuous RRT concluded that there was no evidence for overall superiority of any anticoagulant.It was noted that citrate may have a benefit concerning major bleedings, but that there probably is no meaningful advantage regarding mortality after 28 days nor in preventing formation of clots 61 .
Furthermore, PSM may only adjust for measured baseline covariates, while unmeasured characteristics may remain unbalanced between groups 42 and PSM may not stand in for randomized trials.Nevertheless, as has to be dealt with low patient numbers and regarding possible outcome improvements in sepsis, retrospective data must be taken into consideration.

Interpretation
There was no statistically significant difference in the primary and secondary outcomes, i.e., duration of septic shock nor 30-day mortality, although patients in the RRT + HA cohort seemed to have a slight survival advantage.A post-hoc 90-day survival analysis revealed statistically significant longer survival and lower mortality for patients treated with RRT + HA.Considering some residual differences in baseline characteristics as described above, one has to be careful when interpreting these results as some of those characteristics might interfere with survival in patients treated with RRT + HA.Nevertheless, the most difference in survival probability seems to exist during the first 30 days, as this is where most patients died in the RRT cohort.Factors such as history of tumor disease might reduce overall survival probability, though this should impact survival in the medium rather than the short term.Factors such as coronary heart disease or past myocardial infarction however may interfere with short-term survival.It is conceivable that treatment with RRT + HA during the early phase of septic shock, attempting at preventing the development of immunosuppressive states and empowering the host organism to fight the primary infection may offer some survival benefit for a certain patient collective.The statistical nonsignificance of the present results during the first 30 days may result from the restricted patient cohort, on the other hand, the remaining discrepancies in baseline characteristics may have caused bias in survival analysis.
It would be more compelling to assume that a shorter duration of shock would be easier to demonstrate than lower mortality.However, there are other factors that might come into play, for example the severity of septic shock, or the different phenotypes of septic shock that were described in previous studies 62,63 .There may be undiscovered heterogeneity in therapeutic effects depending on the sepsis phenotype, however, specific analysis of these differences may not be recognized in many studies due to small sample sizes 62 , as is the case for the present investigation.This incoherence of treatment effects and clinical outcomes may also have had an influence on the differences in mortality but not in duration of septic shock seen in this investigation.Future studies on HA should keep in mind the existence of different sepsis phenotypes.
IL-6 levels were decreased in exploratory analysis after initiation of hemoadsorptive therapy, however as IL-6 was not measured in controls an interpretation of IL-6 levels is not possible.
It is currently unclear which patients are most likely to profit from hemoadsorption, although it is conceivable that both patients with gram-negative (with endotoxins) as well as with gram-positive bacterial infection (with translocation of gram-negative species from the digestive tract) may profit from its employment 11 .The use of hemoadsorption may be justified in certain patients; however, there were no statistically significant differences in the primary outcome parameters in this retrospective single center study.This study's patient collective stems from a limited patient population at a large university hospital.Even so, compared to mostly still lower numbers reported in previous studies, the present results provide a considerable contribution to the available literature.

Conclusion
In this retrospective monocenter cohort study, the primary and secondary outcomes of duration of septic shock and 30-day mortality were not statistically significantly different between patients treated with standard of care RRT and patients treated with RRT and hemoadsorption, although patients with hemoadsorption seemed to have a slight survival advantage.
Prospective randomized multicenter studies are warranted to further elucidate the role of simultaneous removal of endotoxins, inflammatory mediators and uremic toxins in ICU patients with septic shock, while care should be taken to account for different sepsis phenotypes.

Fig. 3 .
Fig. 3. Duration of septic shock.Kaplan-Meier survival curves showing durations of septic shock for the matched cohort.The time to event period was defined as the duration from onset of septic shock until resolution of shock.Patients who died before resolution of refractory septic shock occurred were right-censored, and censored cases are marked with vertical bars.(Navy blue, renal replacement therapy (RRT); orange, renal replacement therapy with combined hemoadsorption (RRT + HA).
of view.In this patient cohort, patients received a median minimum heparin dose of 125 (125-423) IU/h and a median maximum heparin dose of 425 (125-545) IU/h during RRT and septic shock.Dialysate.In SLED, dialysate fluids are chosen to match the patient's sodium levels.Available solutions have concentrations of 135, 138, 140 or 145 mmol/L of sodium (Na + ) and standardized bicarbonate levels of 35 mmol/L, which are only adjusted in case of severe acidosis or CO 2 retention.Calcium (Ca 2+ ) concentration is 1.5 mmol/L.Potassium (K + ) concentrations are chosen according to the patient's serum potassium levels, available solutions have concentrations of K + of 2 mmol/L, 3 mmol/L, or 4 mmol/L, respectively.

Table 3 .
Suspected foci and blood culture results.Results are reported in absolute numbers (n).Blood cultures/SerologyBlood cultures returned negative in 14 of 42 cases.Most frequent findings were gram positive (n = 8/42) and gram negative (n = 7/42) bacteria in blood cultures, followed by the combination of both (n = 5/42) and positive virus serology (n = 5/42).Complete findings are reported in Table3.